Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells

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Abstract

The ErbB2 receptor tyrosine kinase is overexpressed in a variety of human tumours. In order to understand the mechanism by which ErbB2 mediates tumour proliferation we have functionally inactivated the receptor using an intracellularly expressed, ER-targeted single-chain antibody (scFV-5R). Inducible expression of scFv-5R in the ErbB2-overexpressing SKBr3 breast tumour cell line leads to loss of plasma membrane localized ErbB2. Simultaneously, the activity of ErbB3, MAP kinase and PKB/Akt decreased dramatically, suggesting that active ErbB2/ErbB3 dimers are necessary for sustained activity of these kinases. Loss of functional ErbB2 caused the SKBr3 tumour cells to accumulate in the G1 phase of the cell cycle. This was a result of reduction in CDK2 activity, which was mediated by a re-distribution of p27Kip1 from sequestering complexes to cyclin E/CDK2 complexes. The level of c-Myc and D-cyclins, proteins involved in p27Kip1 sequestration, decreased in the absence of functional ErbB2. Ectopic expression of c-Myc led to an increase in D cyclin levels, CDK2 activity and resulted in a partial G1 rescue. We propose that c-Myc is a primary effector of ErbB2-mediated oncogenicity and functions to prevent normal p27Kip1 control of cyclinE/CDK2.

Introduction

Normal cellular proliferation is regulated by extracellular factors that trigger signal transduction cascades from surface receptors through cytoplasmic effectors and ultimately control the onset and progression of the cell cycle. Mutations in regulators of this signalling network lead to alterations in cellular proliferation, contributing to cellular malignancy. The ErbB2 receptor tyrosine kinase (RTK) is a key signal transduction molecule that is overexpressed in a variety of human tumours, including breast and ovarian (Berger et al., 1988; Slamon et al., 1987, 1989), where it contributes to poor clinical prognoses (Hynes and Stern, 1994).

There are four ErbB RTKs, EGFR/ErbB1, ErbB2, ErbB3 and ErbB4. Activation of ErbB RTKs is induced by ligand binding and receptor dimerization (Ullrich and Schlessinger, 1990), which leads to autophosphorylation on specific tyrosine residues, creating binding sites for downstream effectors (Pawson and Scott, 1997). No soluble ErbB2 ligand has been identified, however, ligand binding to the other ErbB family members induces heterodimerization and activation of ErbB2. ErbB2 plays a central role since it potentiates and prolongs the signal transduction cascades elicited by ligand activation of the other ErbB RTKs (Graus-Porta et al., 1995). Furthermore, ErbB2 is involved in lateral transmission of signals to ErbB3 and ErbB4 (Graus-Porta et al., 1997; Beerli et al., 1995). ErbB2 has a high basal activity in tumours in which it is overexpressed (Alimandi et al., 1995; DiGiovanna and Stern, 1995) the consequences of which impinge on the other ErbB family members (Graus-Porta et al., 1997). The finding that ErbB3 shows elevated levels of phosphotyrosine in breast tumour cells (Alimandi et al., 1995), implies that ErbB2 contributes to malignant growth by recruiting additional ErbB family member functions, especially that of ErbB3.

In mammalian cells the mechanisms controlling proliferation and division are well characterized (Nigg, 1995; Sherr and Roberts, 1995, 1999). pRb phosphorylation by cyclin dependent kinases (CDKs) in association with their cyclin partners precedes the onset of DNA synthesis. Activity of the CDKs is regulated by various mechanisms including cyclin association, phosphorylation/de-phosphorylation and association with CDK inhibitors (CKIs). Aberrations in cell cycle regulators have been implicated in breast tumour proliferation. Abnormal expression of c-Myc (Escot et al., 1986), the CDK inhibitor (CKI) p27Kip1 and the D and E cyclins has been reported (Catzavelos et al., 1997; Keyomarsi and Pardee, 1993; Schuuring et al., 1992). Despite these alterations, tumour cells must also maintain a critical balance in cell cycle control in order to survive. To date, the mechanism by which oncogenic ErbB2 impinges on proliferation, and the cell cycle components involved, have not been identified. To study ErbB2-dependent proliferation, we have used a strategy for efficient down-regulation of the receptor: intracellular expression of an ErbB2-specific single chain antibody (scFv-5R). Targeting of the intracellularly expressed scFv-5R to the endoplasmic reticulum (ER) causes retention of ErbB2 in this compartment, leading to loss of receptor function (Beerli et al., 1994). While intracellular retention of ErbB2 does not affect growth of tumour cells with low levels of ErbB2 (Graus-Porta et al., 1997; Beerli et al., 1995), it is incompatible with long term growth of ErbB2-overexpressing breast tumour cells (Jannot et al., 1996). Our goal is to identify the crucial cellular components affected by elevated ErbB2 expression and to understand how loss of ErbB2 function impacts on intracellular signalling pathways and on regulators of the cell cycle.

We show here that loss of ErbB2 from the surface of SKBr3 cells caused a decrease in ErbB2 and ErbB3 tyrosine phosphorylation and a dramatic reduction in the activity of the PI3K and Ras/MAP kinase pathways. Parallel with the loss of functional ErbB2, cells accumulated in the G1 phase of the cell cycle. This was the result of a reduction in CDK2 activity by p27Kip1 re-distributing from sequestering complexes to cyclin E/CDK2 complexes. The level of c-Myc protein and mRNA was also decreased in the absence of functional ErbB2. Ectopic expression of c-Myc partially overcame the scFv-5R imposed G1 accumulation, indicating that c-Myc is a primary effector of ErbB2-mediated oncogenicity and functions to mediate p27Kip1 control of cyclinE/CDK2 activity.

Results

Inducible expression of scFv5-R in SKBR-3 cells

We have previously shown that intracellular expression of scFv-5R traps the ErbB2 receptor in the ER. This leads to specific and stable loss of cell surface ErbB2, causing a functional inactivation of the receptor. This technique has allowed us to study the role of ErbB2 in ErbB family signalling (Graus-Porta et al., 1995, 1997; Beerli et al., 1995) and to study its role in transformation (Beerli et al., 1994). Expression of scFv-5R does not affect the growth of tumour cells with low ErbB2 levels, however, its expression is incompatible with long-term growth of ErbB2-overexpressing tumour cells (Jannot et al., 1996). We have shown that loss of plasma membrane ErbB2 and not ER localization of the receptor is responsible for the phenotype (Beerli et al., 1994; Graus-Porta et al., 1995), demonstrating that the cells require active ErbB2 at the plasma membrane for proliferation. To study ErbB2-dependent proliferation, the tetracycline (Tet)-inducible system was used to express scFv-5R in the ErbB2-overexpressing SKBr3 breast tumour cells. A tet-off retroviral vector (Paulus et al., 1996) encoding the tetracycline-regulated, hybrid transactivator (tTA), scFv-5R cloned downstream of the tTa-responsive promoter, and the gene encoding puromycin-resistance, was used to infect SKBr3 cells. A pool of puromycin-resistant cells was selected and revealed down-regulation of ErbB2 in 30–50% of the cells (not shown). Several clones were isolated, all of which responded by down-regulating ErbB2 to various degrees dependent on the amount of scFv-5R expressed upon induction. A clonal derivative (SKBr3-c16) was chosen for further study, based on its low scFv-5R background expression and high inducible expression. scFv-5R expression was monitored over 72 h by Western analysis using a specific polyclonal antiserum (Figure 1a). Expression was noted as early as 12 h (not shown), reaching a maximal between 48 and 72 h. Surface levels of ErbB2 were monitored by flow cytometry and by confocal microscopy, using the ErbB2 specific mAb FSP77 (Harwerth et al., 1992). A decrease in the level of ErbB2 was noted 24 h after scFv-5R induction. Maximal loss of cell surface ErbB2 was reached between 48–72 h (Figure 1b). The kinetics of ErbB2 down-regulation paralleled those observed in the pool and in other selected clones (not shown). This model system enables the study of molecular mechanisms underlying ErbB2-dependent growth in breast cancer cells.

Figure 1
figure1

Inducible scFv-5R expression in SKBr3 cells. (a) SKBR3-c16 cells were induced for the indicated times, cell lysates were prepared and immunoblotted with scFv-5R antiserum. The arrows indicate the under-glycosylated (lower) and glycosylated (upper) forms of scFv-5R. Con indicates control cells. (b) SKBr3-c16 cells were induced for scFv-5R expression and surface levels of ErbB2 were detected on intact cells using mAb FSP77 with a secondary FITC-coupled anti-mouse antibody. Cells were analysed by FACS (first column) and fluorescence microscopy (second column) at the indicated times

ErbB2 down-regulation affects ErbB3 activity

Overexpression of ErbB2 leads to its constitutive activation due to the formation of receptor homodimers (Stern et al., 1988). In many ErbB2-overexpressing tumour cells ErbB3 also has elevated levels of phosphotyrosine (Alimandi et al., 1995; Daly et al., 1997), suggesting that ErbB2/ErbB3 heterodimers are formed in these cells. We examined the activation of both receptors and their ability to couple to downstream effectors following induction of scFv-5R.

ErbB2 retained in the ER/golgi is under-glycosylated, leading to an increase in its electrophoretic mobility (Beerli et al., 1994; Graus-Porta et al., 1995). Uninduced SKBr3-c16 cells express low levels of scFv-5R (Figure 1a, lane 1) which leads to an accumulation of ErbB2 in the ER prior to induction (ErbBER) (Figure 2a, lane 2, upper panel). Despite the rather high levels of ErbBER in un-induced SKBr3-c16 cells, there is no obvious phenotypic effect on the cells very likely because ErbB2 surface expression (ErbB2Sur) was the same as in control cells. The induction of scFv-5R led to a loss in ErbB2Sur (Figure 2a, upper panel) and was paralleled by a decrease in its activity, as determined by the decreased phosphotyrosine content of ErbB2ER (Figure 2a, lower panel).

Figure 2
figure2

Effect of scFv-5R expression on ErbB2, ErbB3 and intracellular signalling pathways. SKBr3-c16 cells were induced for the indicated times, cell lysates were prepared and immunoprecipitated ErbB2 (a) and ErbB3 (b) were immunoblotted with specific antisera (upper panels). After stripping, the membranes were reprobed with anti-phosphotyrosine antibody (lower panels). The arrows in (a) indicate the ER retained (ErbB2ER) and surface (ErbB2Sur) ErbB2. Con indicates control cells. (c) Cell lysates were precipitated using GST-SH2(p85), and immunoblotted with ErbB3 antiserum. (d) Erk1/2 activity was determined by immunoblotting with phospho-p44/42 MAP kinase (Thr202/Tyr204) antiserum (panel 1); PKB activity was measured by reprobing the membrane with phospho-PKB (ser473) antiserum (panel 2). For both blots bands were quantitated and represented as per cent control. The filter was reprobed with a PKB antiserum (panel 3) and MAP kinase antiserum (not shown) to control for loading

Following scFv-5R induction, the level of cell-surface ErbB3, as revealed by flow cytometry (not shown), and total ErbB3 (Figure 2b, upper panel) remained constant. However, the phosphotyrosine content of ErbB3 decreased in parallel with the loss of ErbB2 from the plasma membrane (Figure 2b, lower panel). Compared with the other ErbB receptors, the cytoplasmic tail of ErbB3 contains more potential docking sites for the p85 subunit of PI3K (Fedi et al., 1994; Prigent and Gullick, 1994), allowing it to couple efficiently to this kinase (Beerli and Hynes, 1996). A GST pull-down assay using the SH2 domain of p85, showed that significantly less ErbB3 was precipitated after 72 h scFv-5R induction (Figure 2c, lane 3). This decrease in p85/ErbB3 association paralleled the overall decrease in ErbB3 tyrosine phosphorylation.

Effects of ErbB2 down-regulation on intracellular signalling networks

We next investigated the effects of ErbB2 down-regulation on the kinases ERK1/ERK2, Akt/PKB and p70S6K that are stimulated in response to ErbB receptor activation (Graus-Porta et al., 1995, 1997; Karunagaran et al., 1996; Daly et al., 1999). ERK1/ERK2 were examined using an antiserum specific for the dually phosphorylated, active form of the kinases. There was a 30% drop in ERK1/ERK2 activity after 24 h of scFv-5R induction, with a maximal reduction of 55% reached after 48 h (Figure 2d, upper panel). This result was confirmed by in vitro kinase assays (not shown). We also examined the phosphotyrosine content of Shc, an adapter protein that couples ErbB2 to the MAPK pathway, and found reduced levels after 72 h scFv-5R induction (data not shown).

We examined Akt/PKB, a downstream effector of PI3K (Burgering and Coffer, 1995), using an activation specific antiserum against phospho-S473 (Figure 2d, middle panel). Phosphorylation was reduced by 40% after 24 h scFv-5R induction and by 72 h there was a >90% decrease in the activity of PKB. The activity of p70S6K, another downstream target of PI3K (Chung et al., 1994; Cheatham et al., 1994), decreased slightly after 24 h scFv-5R induction and was reduced by 50% after 72 h (not shown). These results show that loss of cell surface ErbB2 leads to a decrease in the potential of both ErbB2 and ErbB3 to activate multiple signalling pathways.

SKBR-3 cells accumulate in G1 upon down-regulation of ErbB2 by scFv-5R

When induced to express scFv-5R, SKBr3-c16 cells formed fewer and smaller colonies compared with control cells (Figure 3b). BrdU pulse-labelling showed a drop in the percentage of cells undergoing de novo DNA synthesis paralleling the loss of ErbB2Sur (Figure 3a). Flow cytometry indicated that induction of scFv-5R expression led to an accumulation of SKBr3-c16 cells in the G1 phase of the cell cycle. This was noted to varying degrees in the original pool of infected SKBr3 cells as well as in the isolated clonal derivatives and was dependent on the extent of scFv-5R expression (not shown). In SKBr3-c16 the proportion of cells in G1 increased from 64 to 84% over 72 h (Figure 3c, right panel). The G1 accumulation was more pronounced in serum-free medium (90 vs 84% at 72 h) (Figure 3c, left panel), indicating that serum factors can partially compensate for loss of ErbB2. Furthermore, there was no evidence of apoptosis in any of the scFv-5R expressing cultures. In summary, ErbB2 down-regulation appears to restrict the G1 to S progression of SKBr3 cells.

Figure 3
figure3

Analysis of SKBr3-c16 DNA synthesis, proliferation and cell cycle profile following ErbB2 down-regulation. (a) Cells were induced for the indicated times, BrdU was added for the final 4 h, the cells were fixed and stained for incorporated BrdU and DNA content (Hoechst). The data are represented as percentage of BrdU-stained nuclei over total (Hoechst stained) nuclei. (b) SKBr3-c16 cells were plated at low density and cultured in the presence (+) or absence (−) of dox for 9 days. Cells were stained with Giemsa, colonies were counted according to size and expressed as a percentage of the control. (c) Cells were plated at low density in DMEM with (right panels) and without (left panels) serum and induced for the indicated times. Cells were trypsinised, stained with propidium iodide and analysed by flow cytometry. Number in each box represents the percentage of cells in the G1 phase of the cell cycle

ErbB2 controls G1 to S transition via CDK2 phosphorylation of pRb

Hypophosphorylated pRb binds to, and negatively regulates members of the E2F family of transcription factors (Grana et al., 1998). pRb becomes increasingly phosphorylated as cells proceed through G1, leading to the release of these transcription factors and expression of S phase specific genes. Phosphorylation of pRb is controlled by the G1 cyclin-CDKs, the D cyclins associated with CDK4/6 and cyclin E associated with CDK2 (Sherr and Roberts, 1995, 1999).

We examined the phosphorylation status of pRb during the induction of scFv-5R and observed increases in hypophosphorylated pRb as early as 24 h post-induction (Figure 4a). Additionally, there was a decrease in the level of pRb, which is commonly observed in G1 arrested cells (Vlach et al., 1996). Inhibitor studies indicated that this decrease is at least partly MAPK-dependent (R Neve, unpublished data). To determine which of the G1 CDKs were affected by ErbB2 down-regulation, in vitro kinase assays were performed using pRb and Histone H1 as substrates for CDK4 and CDK2, respectively. In multiple, independent assays for CDK4 activity we were unable to detect any significant change following scFv-5R induction. Results from a representative experiment are shown in Figure 4b. Strikingly, by 24 h scFv-5R induction there was a 34% drop in the cyclin E associated kinase activity (Figure 4d), and the total CDK2-associated activity decreased by 90% over 72 h (Figure 4c). This correlates well with the changes in pRb phosphorylation and G1 DNA content which we observed at 24 h, suggesting that the major regulator of pRb phosphorylation is CDK2 in SKBr3 cells.

Figure 4
figure4

Analysis of pRb and G1 cyclin kinase activities after ErbB2 down-regulation. SKBr3-c16 cells were induced for the indicated time and cell lysates were prepared. (a) The phosphorylation status of pRb was determined by immunoblotting. (b) CDK4 kinase activity was measured using GST-Rb as a substrate; (c) CDK2- and (d) cyclinE-kinase activities were measured using Histone H1 as a substrate. The radioactivity in specific bands was determined and the values obtained are shown as per cent (%) control

cyclinE/CDK2 activity is mediated by association with p27Kip1

CDK activity is positively controlled by association with cyclins and negatively controlled by association with inhibitory proteins, the CKIs (Sherr and Roberts, 1995, 1999). Since the reduction in CDK2 activity was not due to a change in cyclin E levels (Figure 5a), we analysed the CKIs. Of the INK4 and Cip/Kip CK1 family members, only p27Kip1 was detectable by immunoblotting in SKBr3 cells either before or after ErbB2 down-regulation. After 24 h scFv-5R induction there was an increase in the amount of p27Kip1 associated with CDK2 (Figure 5c). Immunoprecipitates of Cyclin E showed equivalent elevated levels of complexed p27Kip1 (data not shown). This increase was not a result of increased expression, since p27Kip1 levels remained constant (Figure 5b). p27Kip1 is regulated post-translationally by various mechanisms including sequestration by the D-cyclins (Polyak et al., 1994; Poon et al., 1995). Cyclin D1 was undetectable in SKBr3 cells. There was a strong decrease in the level of both cyclin D2 and cyclin D3 upon ErbB2 down-regulation (Figure 5d,e, lower panels), however, the remaining D-cyclins are apparently sufficient to maintain CDK4 activity (Figure 4b). Corresponding with the loss of D-cyclins, there was a decrease in the amount of p27Kip1 associated with cyclin D immunoprecipitates (Figure 5d,e, upper panels), presumably increasing the availability of uncomplexed p27Kip1 for association with cyclinE/CDK2. In summary, these results suggest that, in SKBr3 cells, ErbB2 controls cyclinE/CDK2 activity by mediating p27Kip1 distribution.

Figure 5
figure5

Analysis of cyclin E expression and cyclin/CDK/CKI complexes upon ErbB2 down-regulation. scFv-5R was induced for the indicated times and cell lysates were analysed. (a) Western blot analysis of cyclin E; (b) Western blot analysis of p27Kip1; (c) CDK2 immunoprecipitates were immunoblotted for p27Kip1 (upper panel) and CDK2 (lower panel). (d) Cyclin D2 immunoprecipitates were immunoblotted for p27Kip1 (upper panel); Western blot analysis of cyclin D2 (lower panel). (e) Cyclin D3 immunoprecipitates were immunoblotted for p27Kip1 (upper panel); Western blot analysis of cyclin D3 (lower panel). Con indicates control cells

c-Myc is pivotal in ErbB2-mediated proliferation

The importance of c-Myc in controlling the activity of G1 cyclin/CDK complexes is well documented (Vlach et al., 1996; Bouchard et al., 1999; Perez-Roger et al., 1999). c-Myc is known to be controlled at multiple levels in many systems (Marcu et al., 1992). Both the PI3 kinase and MAP kinase pathways are implicated in the control of c-Myc cellular levels (Fiddes et al., 1998; Sears et al., 1999). Therefore, having seen a reduction in activity of these pathways following loss of functional ErbB2, we predicted that they would affect c-Myc levels in SKBr3 cells. We observed that c-Myc protein levels (Figure 6a) and mRNA levels (Figure 6b, panel 1) decreased following scFv-5R induction. Similarly, when SKBr3 cells were treated with an ErbB2 inhibitor (PD153035) (Arteaga et al., 1997; Daly et al., 1999), a PI3 kinase inhibitor (LY294002) or a MEK1 inhibitor (PD 98059), each of the inhibitors caused a decrease in c-Myc mRNA levels (Figure 6b, panels 2 and 3). The effect of PD153035 was equivalent to 72 h of ErbB2 down-regulation (Figure 6b, panel 1 vs 3). While LY294002 and PD98059 both decreased c-Myc mRNA, the maximal decrease was observed with a combination of the two (Figure 6b, panel 2), implying that both pathways cooperate in c-Myc regulation in these cells. Furthermore, pulse-chase experiments revealed that the half-life of the c-Myc protein decreased from 60 to 35 min upon 72 h scFv-5R induction (Figure 6c). These results suggest that overexpressed ErbB2 directly influences c-Myc at multiple levels, through PI3 kinase and MAP kinase.

Figure 6
figure6

c-Myc is an effector of ErbB2. (a) c-Myc immunoblot after scFv-5R induction for the indicated times in SKBr3-c16 cells. (b) c-Myc Northern blots; panel 1, after scFv-5R induction in SKBr3-c16 cells for the indicated times; panel 2, SKBr3-c16 cells treated for 6 h with inhibitors for PI3 kinase, L, (LY294002), MEK1, P, (PD98059), or both, L+P; panel 3, SKBr3-c16 cells treated with ErbB inhibitor, EI (PD153035). 18S RNA levels are shown beneath each blot. (c) Protein degradation of c-Myc. SKBr3-c16 cells were taken after 0 h (c16) and 72 h (c16 3d) scFv-5R induction, and a pulse chase was performed as described in Materials and methods. Chase times, in minutes, are shown above the lanes. Bands were quantitated and are shown as per cent control below each panel. (d) scFv-5R was induced concurrently with AdMyc infection. Cell lysates were analysed for: panel 1, c-Myc (a), G1 DNA content (b) and G1 DNA content in control AdLacZ infected cells (c); panel 2, CDK2 kinase activity (a), quantitated below (b); panel 3, association of p27Kip1 with CDK2; panel 4, pRb phosphorylation; panel 5, cyclin D3 levels

In order to test if ectopic expression of c-Myc could overcome the scFv-5R mediated G1 block, cells were infected with a recombinant c-Myc-expressing adenovirus (AdMyc). Expression of c-Myc following 72 h scFv-5R induction was unable to push cells out of G1 (not shown). However, concurrent AdMyc infection and scFv-5R induction (Figure 6d, panel 1a) delayed the accumulation of cells in G1 (Figure 6d, panel 1b). Using LacZ adenovirus (AdLacZ) as a control, equivalent amounts of AdLacZ had no effect on the G1 content (Figure 6d, panel 1c) or other cell cycle components affected by the AdMyc virus (not shown). AdMyc expression also prevented p27Kip1 association with cyclin E/CDK2 (Figure 6d, panel 3), increased pRb phosphorylation and stabilized pRb protein levels (Figure 6d, panel 4) while CDK2 kinase activity remained high (Figure 6d, panel 2a, quantitated in 2b). Consistent with the elevation of CDK2 activity, levels of cyclin D2 (not shown) and cyclin D3 (Figure 6d, panel 5) were increased in AdMyc infected cells for the first 48 h of scFv-5R induction. At 72 h, cells began to accumulate in G1 and cyclin D3 (Figure 6d, panel 5) levels decreased. The level of c-Myc protein did not decrease in AdMyc infected cells as c-Myc is under control of an exogenous promoter, therefore bypassing endogenous transcriptional control. In conclusion, these results show that ErbB2 requires c-Myc to regulate CDK2 activity, however, continuous and timely upstream signals emanating from ErbB2 appear to be required to sustain proliferative potential.

Discussion

In this paper we have examined the role of ErbB2 in the proliferation of tumour cells overexpressing this receptor. Our goal was to understand how this transmembrane receptor impinges on the cell cycle, allowing tumour cells with multiple molecular alterations to progress in an orderly manner through the cell cycle. Our results, and data from other studies employing different methods to inhibit ErbB2 (Czubayko et al., 1997; Ebbinghaus et al., 1993; Juhl et al., 1997), show that ErbB2 plays a central role in the proliferation of these tumour cells. We show here the mechanism underlying this growth inhibition. The inhibition of proliferation is due to a G1 accumulation of the cells. Furthermore, it appears that ErbB2 and ErbB3 function together to stimulate signalling networks that result in proliferation. ErbB2 regulates early cell cycle progression by a process that principally involves p27Kip1. The results indicate that the loss of functional ErbB2 leads to a decrease in c-Myc and D cyclins releasing sequestered p27Kip1 to complex with, and inhibit CDK2 activity. Ectopic expression of c-Myc reverses this process with a concomitant increase in cyclin D levels. This suggests that c-Myc is an essential target of oncogenic ErbB2.

Human breast tumours overexpressing ErbB2 display elevated levels of phosphotyrosine on ErbB2 and ErbB3. Since ErbB3 has impaired kinase activity (Carraway and Cantley, 1994), ErbB2 is required to laterally transmit activating signals to ErbB3 (Graus-Porta et al., 1997). It was recently shown in a transgenic model of Neu-induced mammary cancer that tumour progression was associated with a dramatic increase in ErbB3 tyrosine phosphorylation (Siegel et al., 1999). Moreover, the ErbB2/ErbB3 heterodimer appears to be the most potent ErbB signalling complex in terms of in vitro growth and transformation (Alimandi et al., 1995; Pinkas-Kramarski et al., 1996; Zhang et al., 1996; Waterman et al., 1999). ErbB3 couples well to the PI3K pathway due to the multiple binding sites for p85 (Fedi et al., 1994; Prigent and Gullick, 1994). The gene encoding the p110α catalytic subunit of PI3K is amplified in many ovarian tumours and cell lines, some of which do not express ErbB3 (Shayesteh et al., 1999), implicating this pathway in tumorigenesis. Our results show that intracellular retention of ErbB2 leads to a dramatic decrease in ErbB3 phosphorylation and its association with PI3 kinase. Thus, we favour the hypothesis that the ErbB2/ErbB3 heterodimer is responsible for transformation in SKBr3 cells due to its ability to promote strong activation of cytoplasmic signalling pathways.

Our results show that D cyclins and c-Myc are important nuclear targets of the ErbB2/ErbB3 dimer. Down-regulation of ErbB2/ErbB3 signalling as well as inhibitors for ErbB, PI3 kinase and MEK1 all caused a decrease in c-Myc RNA levels. D cyclin and c-Myc protein levels also decreased upon inhibitor treatment (not shown). Previous reports show that activation of the MAP kinase pathway stabilizes the c-Myc protein (Sears et al., 1999) and increases D cyclin transcription (Meyerson and Harlow, 1994). Our results show that high levels of ErbB2/ErbB3 signalling enhance the stability of c-Myc protein. Furthermore, decreased signalling of the PI3 kinase-PKB pathway activates GSK3β that phosphorylates and targets D cyclins for degradation (Diehl et al., 1998; Cheng et al., 1999). The regulation of D cyclins and c-Myc by ErbB2/ErbB3 dimers is due to multiple effects at the level of transcription, protein stability and degradation. Presently there is no clinical data correlating ErbB2 and c-Myc expression levels. However, our results indicate such a link may exist.

Cyclin D-dependent kinase complexes, as well as the c-Myc transcription factor, play major roles in the regulation of p27Kip1 sequestration (Sherr and Roberts, 1995, 1999; Vlach et al., 1996; Bouchard et al., 1999; Perez-Roger et al., 1999). Since the level of the CKI p27Kip1 is not altered by loss of ErbB2 function in SKBr3 cells, the decrease in D cyclins and c-Myc following loss of ErbB2 signalling, very likely results in a re-distribution of p27Kip1 onto cyclin E/CDK2 complexes, leading to inhibition of kinase activity. Simultaneous induction of scFv-5R and ectopic expression of c-Myc prevented the G1 block and p27Kip1/CDK2 association for the first 48 h of AdMyc infection. Accordingly, the level of CDK2 activity remained high during this time. Ectopic c-Myc expression increased cyclin D2 and D3 levels in SKBr3 cells, suggesting these as targets of c-Myc, as recently shown in other cell lines (Bouchard et al., 1999; Perez-Roger et al., 1999). However, despite the fact that c-Myc levels were still elevated at 72 h, the level of cyclin D3 dropped, which may in part account for the appearance of p27Kip1 in CDK2 complexes and the increase of cells in G1. The drop in D cyclin levels is probably due to the decrease in MAP kinase and PI3K activities seen at 72 h, which as discussed above, are required for D cyclin expression and stability. This may also explain why ectopic c-Myc expression, 72 h after scFv-5R induction, was unable to induce an exit from G1. These results show that elevated levels of c-Myc alone cannot fully compensate for the proliferative signals emanating from oncogenic ErbB2. In the light of numerous publications showing that cooperation between c-Myc and Ras is essential for transformation of primary cells (Kauffmann-Zeh et al., 1997; Leone et al., 1997; Yancopoulos et al., 1985), these results are not unexpected. Hence, we conclude that ErbB2 overexpression affects the cell cycle at multiple critical points leading to a regulated upregulation of normal cellular proliferation.

In normal cells there is a complex network of feedback loops maintaining a balance between positive and negative regulators of cell cycle progression. Despite the fact that many cell cycle regulators are altered (Catzavelos et al., 1997; Keyomarsi and Pardee, 1993; Spencer and Groudine, 1991), tumour cells maintain homeostasis between growth promoting and growth inhibiting processes. Induction of scFv-5R for 24 h, a time when there were still significant levels of cell surface ErbB2, disrupted the activity of the PI3K and Ras/MAP kinase pathways and led to an increase of cells in G1. This suggests that the balance between signals promoting and inhibiting proliferation is finely tuned and a minor disturbance leads to perturbation of proliferation. Importantly, this implies that drugs that target the receptor may have clinical efficacy without totally blocking ErbB2 function.

Materials and methods

Antibodies and reagents

Anti-CDK2 (M2), anti-CDK4 (C-22), anti-cyclin D2 (C-17), anti-cyclin D3 (C-16), anti-cyclin E (HE12), anti-ErbB3 (C17) and GST-pRb were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-pRb (G3-245) was from Pharmingen. Anti-Akt (PKB), anti-phospho-Akt (Ser473) and anti-phospho-p44/42 MAP Kinase (Thr202/Tyr204) were from New England Biolabs. Anti p27Kip1 was from Transduction Laboratories and anti-c-Myc (9E10, affinity purified) was a kind gift from Trevor Littlewood. Myc antibody C-33 (Santa Cruz Biotechnology, Inc) was used for immunoprecipitations. Anti-ErbB2 mAb FSP77 and anti-scFv-5R polyclonal rabbit serum have been described (Wels et al., 1992; Harwerth et al., 1992). Histone H1 was from Boehringer. Doxycycline (dox) and puromycin were from Sigma. Inhibitors PD153035 (4 μM), LY294002 (50 μM) (Calbiochem) and PD98059 (50 μM) (New England Biolabs, MA, USA) were stored as 1000× concentrated stock solutions in DMSO.

Measurement of protein stability

Cells were labelled in vivo with 200 μCi L 35S-methionine/cysteine PRO-MIXTM from Amersham at a ratio of 3×105 cells/ml for 2 h. After labelling, cells were immediately washed twice with DMEM containing 5 mM L-methionine, 3 mM L-cysteine and then incubated in the same medium for the indicated chase times. Cells were harvested and c-Myc was immunoprecipitated from 106 cells for each sample, as described below. To confirm that equal amounts of labelled protein was analysed for each sample, aliquots of the immunoprecipitation supernatants were analysed by SDS–PAGE followed by Western blot. Labelled c-Myc was visualized by autoradiography and quantitated using a phosphoimager.

Northern analysis

Total RNA was isolated using the acid-phenol method. After electrophoresis on a formaldehyde-agarose gel and transfer to a nitrocellulose membrane, mRNA expression levels were evaluated using a RediPrime (Amersham) labelled cDNA probe corresponding to the complete c-Myc human coding region. Hybridization was performed overnight at 65°C followed by four washes in 0.1% SDS/2×SSC at 65°C. Bands were visualized and quantitated by phosphoimager. Cells were treated for 6 h with specific kinase inhibitors prior to RNA extraction.

Cell culture, transfections and infection

The SKBr3 breast carcinoma cell line was maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal calf serum (FCS). The inducible scFv-5R construct was introduced after cloning into the pBSTR1-Puro retroviral vector (Paulus et al., 1996). High-titer retroviral supernatants were generated by transiently transfecting pBSTR1-Puro-scFv-5R plasmid DNA into Bing packaging cells. SKBr3 cells were infected and selected in 2 μg/ml Puromycin. Individual clones were tested for high inducible levels of scFv-5R. A clone, SKBr3-c16, was selected and grown in medium supplemented with 2 ng/ml dox to suppress scFv-5R expression. To induce scFv-5R, 106 cells were plated on a 10 cm dish, washed once in PBS, fresh medium was added and cells were harvested at the indicated times. Control cells were infected with empty pBSTR1-puro retroviral vector.

Recombinant adenovirus generation and infection

To construct pAdlox-c-Myc, a BamHI–EcoRI fragment containing the human c-Myc cDNA (obtained from B Amati) was isolated and cloned into pAdlox digested with BamHI-EcoRI. Recombinant c-Myc adenovirus was generated by co-transfecting SfiI-linearised pAdlox-c-Myc plasmid DNA and Ψ5 adenovirus DNA in 293-CRE8 cells as previously described (Hardy et al., 1997). Recombinant adenoviruses were selected by serial infection of 293-CRE8 cells and subsequently amplified in 293 cells. Adenoviruses were purified by CsCl-density gradient centrifugation, dialysed against HBS buffer (10 mM HEPES, pH 7.2, 100 mM NaCl, 1 mM MgCl2) and stored at −20°C. Infections of cells were carried out for the indicated times.

Cell cycle analysis

Trypsinized cells were resuspended in PI staining buffer (1 mM sodium citrate, pH 4.0, 1.5 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% NP40, 4 μg/ml propidium iodide, 80 μg/ml RNase A in PBS), placed on ice for 1 h, then a cell cycle analysis was perfomed on a Becton Dickinson FACScan Flow Cytometer. Surface ErbB2 levels were detected by immunostaining with mAb FSP77 which binds the extracellular domain as described (Beerli et al., 1994; Harwerth et al., 1992).

Cell lysis, immunoprecipitation and immunoblots

Cells were lysed in NP40 lysis buffer (50 mM HEPES pH 7.4, 1% NP40, 150 mM NaCl, 25 mM β-glycerol phosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 10 μg/ml leupeptin and aprotinin, 1 mM PMSF) and cell debris was pelleted at 10 000 g for 30 min. Protein concentration was determined using the Bradford Biorad kit. Proteins were resolved on 7.5–15% SDS–PAGE gels, blotted onto PVDF and detected by ECL.

Rb and Histone H1 kinase assays

Rb kinase assays; Cell lysates were prepared in RB lysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween-20, 10% glycerol, 80 mM β-glycerol phosphate, 1 mM NaF, 0.1 mM sodium orthovanadate, 1 mM PMSF, 10 μg/ml leupeptin and aprotinin) and immunoprecipitated with antibodies and protein A sepharose. After washing, the immunoprecipitates were incubated for 30–60 min at 30°C in 30 μl kinase buffer (50 mM HEPES-KOH pH 7.5, 10 mM MgCl2, 1 mM DTT, 2.5 mM EGTA, 0.1 mM β-glycerol phosphate, 1 mM NaF and 0.1 mM sodium orthovanadate) supplemented with 5 μg GST-Rb, 20 μM ATP and 5 μCi of [γ-32P]ATP. The reaction was stopped by addition of SDS sample buffer. The samples were boiled for 5 min, resolved by SDS–10%PAGE and visualized with a phosphoimager. Histone H1 assays were identical to the Rb assays except lysates were made using NP40 lysis buffer and Histone H1 was used as a substrate.

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Acknowledgements

Richard Neve was partially supported by a grant from the Basel Cancer League. Heidi Lane and John Daly were partially supported by grants from the Swiss Cancer League. We thank Thomas Holbro, Ulrich Müller, Chris Benz, Bruno Amati, Trevor Littlewood, Peter Traxler, Alain Marti, and Matthias Senften for helpful discussions and reagents and Sinisa Volarevic for invaluable input.

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Correspondence to Nancy E Hynes.

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Keywords

  • single chain antibody
  • p27kip1
  • c-Myc
  • PI3 kinase
  • MAP kinase

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